1 Introduction

MuGam is a orthologue of eukaryotic proteins Ku70/Ku80, which are involved in recognizing double-strand DNA prior to repair, as well as DNA transposition. In Haemophilus influenzae, Gam (HiGam) inhibits digestion of Mu phage DNA by nucleases (d’Adda di Fagagna, Weller, Doherty, et al., 2003).

The aim of this report is to characterise HiGam by processing the data obtained from the following experiments:

  • Polymerase chain reaction (PCR)
  • Electrophoretic mobility shift assay (EMSA)
  • Size-exclusion chromatography (SEC)
  • Analytical ultracentrifugation (AUC) ???

2 Requirements

Required software and packages are outlined in the README.

3 Preparing Data


4 Analysis

4.1 PCR

Diagnostic PCR was used to amplify the H. influenzae homologues, and to test for their transcription relative to the Genomic DNA (gDNA) control. Amplicon sizes were established using DNA ladder molecular weight (MW) standards. Results identified the transcription of HiA, HiB, HiGam, HiA-HiB and HiB-HiGam in H. influenzae. Figure @(fig:pcr-fig) shows

\[\Large \color{#8D9FCA} y \color{black} = \color{#E789C2} m \color{#66c2a4} x \color{black} + \color{#FFD82F} c\]


4.2 Electrophoretic Mobility Shift Assay (EMSA) & Binding Affinity

Electrophoretic mobility shift assay (EMSA) was used to examine whether Im9-HiGam–DNA had an affinity for closed circular DNA or linear DNA. Im9-HiGam was observed to bind preferentially to linear DNA with a KD of approximately 0.55 (4.1). Results estimate Im9-HiGam has a binding dimeric footprint of 22 base pairs.

(fig:emsa-fig)

Figure 4.1: (fig:emsa-fig)


4.3 Size Exclusion Chromatography (SEC)

Size exclusion chromatography (SEC) was used to explore the molecular weight and oligomeric state of Im9-HiGam. 7 chromatograms of known MW were plotted and a linear regression was performed to determine the relationship between elution volume and molecular weight. The SEC data showed a peak for both wavelengths, with a MW of approximately 58853.86 Daltons for A254 and 77703.42 Daltons for A280. This gives Im9-HiGam has an estimated dimeric footprint of approximately 28.56 base pairs.

4.3.1

SEC analysis

SEC Standards

SEC dataset

Table 4.1: SEC Absorbencies for a254 & a280
Volume a254 a280
0.00 0.10 0.18
0.02 0.20 0.18
0.05 0.07 -0.01
0.07 -0.04 -0.04
0.10 -0.02 -0.20
0.12 -0.11 0.10
0.14 0.07 -0.09
0.17 -0.03 0.06
0.19 -0.06 -0.36
0.22 0.07 -0.15
0.24 0.14 -0.33
0.26 -0.12 -0.10
0.29 0.19 0.14
0.31 0.02 0.04
0.34 0.05 0.12
0.36 0.03 0.19
0.38 -0.02 -0.32
0.41 0.12 0.21
0.43 -0.13 0.01
0.46 0.08 -0.07
0.48 -0.11 0.15
0.50 -0.06 -0.03
0.53 -0.11 -0.02
0.55 0.01 -0.02
0.58 -0.15 -0.04
0.60 0.10 -0.03
0.62 0.13 -0.12
0.65 -0.17 0.32
0.67 0.05 0.04
0.70 0.19 0.27
0.72 0.12 -0.21
0.74 0.20 -0.25
0.77 0.08 0.06
0.79 -0.14 0.18
0.82 -0.03 0.04
0.84 -0.17 -0.13
0.86 0.10 0.08
0.89 0.07 -0.04
0.91 0.02 0.35
0.94 0.16 -0.02
0.96 0.00 0.09
0.98 0.03 0.01
1.01 -0.10 0.12
1.03 -0.16 -0.23
1.06 0.13 -0.09
1.08 -0.19 -0.04
1.11 0.11 -0.14
1.13 0.03 0.00
1.15 0.04 -0.05
1.18 -0.06 0.01
1.20 -0.18 0.02
1.23 0.05 0.15
1.25 0.01 0.02
1.27 -0.14 -0.27
1.30 0.15 -0.19
1.32 -0.17 -0.29
1.35 -0.02 -0.18
1.37 0.14 -0.15
1.39 0.02 0.10
1.42 -0.06 -0.27
1.44 0.01 0.03
1.47 -0.13 0.05
1.49 0.07 -0.32
1.51 -0.07 -0.21
1.54 -0.15 -0.04
1.56 0.01 -0.02
1.59 -0.01 0.11
1.61 -0.13 -0.16
1.63 0.03 0.19
1.66 0.13 0.19
1.68 -0.18 -0.09
1.71 0.03 0.07
1.73 0.15 -0.02
1.75 0.03 0.14
1.78 -0.01 0.18
1.80 0.14 -0.19
1.83 0.11 0.26
1.85 -0.15 -0.14
1.87 -0.17 -0.24
1.90 0.11 -0.03
1.92 -0.21 0.17
1.95 0.12 0.29
1.97 0.06 -0.28
1.99 0.19 -0.03
2.02 0.15 -0.06
2.04 -0.10 0.21
2.07 -0.21 -0.19
2.09 0.06 0.08
2.11 0.05 -0.02
2.14 0.17 -0.34
2.16 -0.13 -0.24
2.19 0.17 0.10
2.21 -0.10 0.08
2.23 0.06 -0.04
2.26 -0.14 0.00
2.28 0.09 0.10
2.31 -0.04 0.18
2.33 0.03 0.12
2.35 -0.03 0.21
2.38 -0.19 0.06
2.40 0.19 0.33
2.43 -0.06 0.24
2.45 0.12 0.28
2.47 -0.12 0.07
2.50 0.16 0.19
2.52 -0.14 0.21
2.55 0.17 0.02
2.57 -0.07 0.12
2.59 0.17 0.01
2.62 0.14 -0.06
2.64 0.10 0.18
2.67 -0.05 0.17
2.69 0.10 0.03
2.71 0.11 0.24
2.74 -0.10 -0.08
2.76 0.03 -0.03
2.79 0.05 -0.01
2.81 -0.13 0.33
2.83 0.08 0.03
2.86 0.15 -0.02
2.88 -0.02 0.32
2.91 -0.20 0.35
2.93 0.00 -0.07
2.95 0.07 0.18
2.98 -0.12 0.06
3.00 -0.01 0.10
3.03 -0.03 0.06
3.05 -0.14 -0.16
3.08 -0.02 0.13
3.10 0.00 -0.11
3.12 -0.10 -0.20
3.15 -0.13 -0.07
3.17 0.02 -0.13
3.20 0.13 0.21
3.22 0.19 -0.18
3.24 0.14 -0.08
3.27 0.18 0.24
3.29 -0.16 -0.31
3.32 0.05 -0.23
3.34 -0.02 0.05
3.36 -0.02 -0.11
3.39 -0.02 0.12
3.41 0.02 -0.21
3.44 0.04 0.12
3.46 -0.10 0.01
3.48 0.01 0.03
3.51 0.09 0.11
3.53 0.09 -0.03
3.56 0.12 -0.08
3.58 0.19 -0.06
3.60 0.10 0.06
3.63 -0.01 -0.12
3.65 0.19 -0.02
3.68 -0.11 -0.16
3.70 -0.06 -0.08
3.72 -0.03 -0.39
3.75 0.00 -0.11
3.77 -0.07 0.00
3.80 0.11 -0.16
3.82 0.13 -0.07
3.84 0.10 -0.22
3.87 -0.16 -0.20
3.89 0.16 -0.08
3.92 0.06 0.32
3.94 0.11 0.13
3.96 -0.05 -0.12
3.99 -0.17 -0.18
4.01 0.11 -0.04
4.04 -0.02 -0.31
4.06 -0.05 -0.02
4.08 0.11 0.02
4.11 0.13 -0.32
4.13 0.15 -0.16
4.16 0.11 -0.28
4.18 0.03 -0.03
4.20 0.06 0.06
4.23 -0.01 0.15
4.25 -0.03 0.16
4.28 -0.01 0.15
4.30 -0.15 0.07
4.32 0.20 -0.01
4.35 -0.15 -0.15
4.37 -0.02 0.12
4.40 0.16 0.10
4.42 -0.13 -0.23
4.44 -0.05 -0.13
4.47 -0.15 -0.29
4.49 -0.03 0.03
4.52 -0.01 0.07
4.54 -0.03 -0.19
4.56 0.12 -0.04
4.59 -0.20 -0.01
4.61 0.12 0.27
4.64 0.04 -0.21
4.66 -0.12 -0.18
4.68 0.03 -0.15
4.71 -0.08 -0.05
4.73 -0.06 0.09
4.76 -0.06 0.12
4.78 -0.19 -0.01
4.80 0.02 -0.35
4.83 0.02 0.33
4.85 0.09 0.25
4.88 -0.14 -0.10
4.90 0.15 0.18
4.92 0.15 0.12
4.95 -0.10 0.02
4.97 -0.03 -0.07
5.00 -0.05 -0.01
5.02 -0.13 -0.03
5.05 0.04 0.23
5.07 0.09 -0.07
5.09 0.09 -0.06
5.12 0.02 0.04
5.14 -0.15 -0.15
5.17 -0.11 0.02
5.19 -0.03 0.31
5.21 -0.20 0.01
5.24 0.06 -0.15
5.26 0.00 -0.24
5.29 0.09 0.21
5.31 -0.08 0.05
5.33 0.07 -0.12
5.36 0.11 0.09
5.38 -0.12 -0.06
5.41 -0.03 -0.14
5.43 -0.05 -0.03
5.45 0.13 -0.18
5.48 -0.06 -0.03
5.50 0.20 0.15
5.53 -0.05 0.20
5.55 0.05 -0.08
5.57 0.14 0.05
5.60 0.06 -0.12
5.62 -0.12 0.18
5.65 -0.16 0.01
5.67 -0.11 -0.20
5.69 -0.16 -0.28
5.72 -0.11 0.06
5.74 -0.16 -0.20
5.77 -0.07 -0.07
5.79 0.01 -0.01
5.81 0.16 -0.07
5.84 -0.12 0.06
5.86 0.12 0.12
5.89 -0.07 0.19
5.91 0.02 0.27
5.93 -0.10 -0.02
5.96 -0.12 -0.05
5.98 0.18 0.28
6.01 -0.15 0.01
6.03 0.12 -0.07
6.05 -0.16 0.24
6.08 0.02 0.06
6.10 -0.01 0.05
6.13 -0.08 0.25
6.15 0.09 0.24
6.17 -0.01 0.27
6.20 -0.03 -0.16
6.22 0.07 -0.32
6.25 -0.19 -0.04
6.27 -0.19 -0.22
6.29 -0.08 0.22
6.32 0.17 -0.16
6.34 -0.08 0.01
6.37 -0.11 -0.30
6.39 -0.12 0.03
6.41 0.03 -0.29
6.44 -0.12 0.31
6.46 -0.08 0.01
6.49 0.13 0.21
6.51 0.05 -0.06
6.53 0.16 -0.06
6.56 -0.13 -0.12
6.58 0.17 0.33
6.61 0.13 0.00
6.63 0.12 -0.09
6.65 -0.18 -0.06
6.68 0.21 0.33
6.70 0.12 0.31
6.73 -0.11 0.00
6.75 -0.17 0.00
6.77 0.16 0.23
6.80 -0.03 -0.05
6.82 -0.09 0.27
6.85 0.18 0.01
6.87 0.17 -0.27
6.89 -0.09 0.11
6.92 0.01 -0.17
6.94 -0.17 -0.02
6.97 0.19 0.01
6.99 -0.06 0.29
7.02 0.01 0.04
7.04 0.11 -0.17
7.06 0.02 -0.33
7.09 -0.01 -0.15
7.11 -0.07 -0.30
7.14 -0.05 -0.26
7.16 -0.05 -0.04
7.18 -0.12 -0.17
7.21 0.14 -0.10
7.23 -0.08 -0.08
7.26 -0.16 0.21
7.28 -0.03 -0.12
7.30 -0.16 0.13
7.33 0.18 0.12
7.35 -0.19 0.14
7.38 0.00 0.17
7.40 0.08 0.23
7.42 -0.18 0.14
7.45 -0.03 -0.22
7.47 0.04 0.21
7.50 -0.04 0.07
7.52 -0.14 -0.08
7.54 -0.08 0.05
7.57 0.16 0.02
7.59 0.04 -0.08
7.62 0.01 0.27
7.64 -0.17 -0.05
7.66 0.06 0.24
7.69 0.05 -0.05
7.71 0.15 -0.05
7.74 -0.11 0.03
7.76 0.17 0.22
7.78 -0.18 0.31
7.81 -0.04 0.25
7.83 -0.08 -0.34
7.86 -0.19 -0.37
7.88 -0.08 -0.05
7.90 0.17 0.11
7.93 -0.20 0.03
7.95 -0.01 0.22
7.98 0.18 -0.14
8.00 -0.12 -0.06
8.02 0.08 -0.31
8.05 -0.04 -0.23
8.07 0.16 0.01
8.10 -0.18 0.17
8.12 -0.16 -0.13
8.14 0.06 0.21
8.17 0.08 -0.13
8.19 -0.04 0.01
8.22 -0.04 -0.16
8.24 0.15 -0.15
8.26 -0.18 -0.15
8.29 0.06 -0.05
8.31 -0.21 0.24
8.34 0.12 0.03
8.36 0.02 -0.22
8.38 0.10 0.06
8.41 -0.09 0.28
8.43 -0.18 -0.04
8.46 0.03 -0.05
8.48 0.06 -0.18
8.50 0.10 0.07
8.53 0.08 0.31
8.55 -0.03 0.06
8.58 0.19 0.08
8.60 -0.08 0.01
8.62 -0.03 -0.17
8.65 -0.10 -0.12
8.67 -0.17 0.31
8.70 -0.13 -0.17
8.72 0.07 0.22
8.74 -0.08 0.20
8.77 0.22 0.10
8.79 0.12 -0.21
8.82 -0.11 0.07
8.84 -0.02 0.24
8.86 -0.19 0.18
8.89 0.16 -0.08
8.91 0.02 -0.13
8.94 0.17 0.24
8.96 -0.12 0.16
8.98 -0.10 0.13
9.01 -0.12 0.13
9.03 0.01 -0.09
9.06 -0.02 0.15
9.08 -0.10 0.25
9.11 -0.06 0.08
9.13 -0.01 -0.08
9.15 0.05 -0.07
9.18 0.16 0.04
9.20 0.11 -0.36
9.23 0.05 0.01
9.25 -0.10 -0.09
9.27 0.15 -0.06
9.30 -0.19 -0.15
9.32 -0.10 0.21
9.35 -0.13 -0.07
9.37 0.06 -0.21
9.39 0.09 -0.03
9.42 0.19 0.04
9.44 0.15 -0.18
9.47 -0.19 0.03
9.49 0.13 -0.01
9.51 0.09 -0.33
9.54 0.14 0.02
9.56 0.17 0.05
9.59 0.01 -0.17
9.61 0.01 -0.26
9.63 -0.09 0.08
9.66 -0.03 0.16
9.68 -0.06 -0.02
9.71 0.07 -0.01
9.73 -0.04 -0.15
9.75 0.05 0.01
9.78 -0.10 -0.14
9.80 0.00 -0.15
9.83 0.02 -0.05
9.85 0.10 0.06
9.87 -0.07 0.25
9.90 -0.01 0.02
9.92 0.11 -0.01
9.95 -0.16 0.01
9.97 -0.09 -0.03
9.99 0.18 -0.21
10.02 -0.01 -0.05
10.04 0.18 0.08
10.07 0.04 0.11
10.09 -0.12 0.05
10.11 -0.05 0.04
10.14 -0.13 -0.30
10.16 0.08 -0.30
10.19 0.06 -0.33
10.21 0.00 -0.12
10.23 0.16 -0.02
10.26 0.04 0.22
10.28 -0.18 -0.24
10.31 0.00 -0.06
10.33 0.03 -0.16
10.35 -0.13 0.18
10.38 0.14 0.28
10.40 -0.10 0.04
10.43 -0.08 0.37
10.45 0.15 0.20
10.47 0.00 0.00
10.50 0.01 -0.37
10.52 -0.17 -0.14
10.55 0.10 0.13
10.57 0.14 -0.01
10.59 0.06 -0.30
10.62 0.14 -0.20
10.64 0.09 -0.26
10.67 -0.02 0.31
10.69 0.06 -0.29
10.71 -0.04 0.06
10.74 0.17 0.05
10.76 -0.10 0.22
10.79 0.15 0.03
10.81 0.08 0.18
10.83 0.20 -0.02
10.86 0.05 -0.23
10.88 -0.11 -0.01
10.91 -0.10 -0.27
10.93 0.06 -0.22
10.95 -0.07 -0.34
10.98 0.07 -0.30
11.00 -0.16 0.17
11.03 0.15 0.03
11.05 -0.19 -0.06
11.08 -0.09 0.00
11.10 0.05 -0.13
11.12 -0.04 0.10
11.15 0.12 0.07
11.17 -0.15 0.08
11.20 0.08 0.16
11.22 0.18 -0.29
11.24 -0.13 0.29
11.27 0.00 0.35
11.29 0.14 0.15
11.32 0.17 0.11
11.34 0.15 -0.15
11.36 -0.04 0.12
11.39 0.04 -0.15
11.41 0.02 0.03
11.44 -0.09 0.09
11.46 -0.19 0.25
11.48 0.02 -0.25
11.51 0.08 0.15
11.53 0.14 -0.04
11.56 -0.04 -0.19
11.58 0.04 -0.17
11.60 -0.08 -0.08
11.63 0.03 -0.09
11.65 -0.14 0.02
11.68 0.01 0.02
11.70 0.09 0.17
11.72 0.04 0.01
11.75 0.18 0.16
11.77 -0.07 0.12
11.80 -0.14 0.03
11.82 -0.09 -0.12
11.84 0.05 -0.20
11.87 -0.02 -0.04
11.89 0.12 -0.06
11.92 -0.18 0.11
11.94 -0.07 -0.04
11.96 -0.05 -0.04
11.99 0.06 -0.16
12.01 0.05 0.28
12.04 0.21 -0.04
12.06 -0.03 0.23
12.08 0.19 0.03
12.11 0.14 0.20
12.13 -0.01 0.11
12.16 -0.05 0.07
12.18 0.13 -0.08
12.20 0.10 -0.01
12.23 0.10 -0.03
12.25 -0.17 -0.21
12.28 -0.19 -0.25
12.30 -0.05 -0.33
12.32 0.03 -0.24
12.35 0.04 -0.08
12.37 -0.01 0.01
12.40 -0.02 0.06
12.42 -0.01 0.01
12.44 -0.03 -0.07
12.47 -0.12 -0.02
12.49 0.16 0.18
12.52 0.08 0.05
12.54 -0.01 -0.08
12.56 0.21 -0.15
12.59 -0.01 0.01
12.61 -0.12 -0.11
12.64 -0.09 0.09
12.66 -0.19 0.21
12.68 0.11 0.04
12.71 -0.09 -0.01
12.73 0.11 -0.29
12.76 0.19 0.03
12.78 0.04 0.09
12.80 0.19 -0.02
12.83 0.13 0.02
12.85 0.08 0.09
12.88 -0.01 0.12
12.90 -0.07 0.05
12.92 0.13 -0.12
12.95 -0.12 -0.18
12.97 0.08 0.25
13.00 0.00 -0.16
13.02 -0.01 -0.13
13.05 -0.12 0.27
13.07 -0.16 0.07
13.09 -0.03 0.13
13.12 -0.14 0.21
13.14 -0.07 0.09
13.17 0.07 0.33
13.19 0.04 0.12
13.21 -0.10 -0.23
13.24 -0.14 0.29
13.26 0.04 0.12
13.29 0.07 0.12
13.31 0.10 0.29
13.33 -0.09 0.11
13.36 -0.17 0.13
13.38 0.11 -0.26
13.41 -0.12 -0.03
13.43 -0.03 0.00
13.45 0.14 0.04
13.48 0.11 -0.22
13.50 0.13 0.27
13.53 -0.04 0.10
13.55 -0.13 -0.22
13.57 0.19 -0.10
13.60 -0.16 0.05
13.62 0.10 0.17
13.65 0.10 -0.16
13.67 0.11 0.21
13.69 -0.18 0.11
13.72 0.17 0.05
13.74 0.17 0.38
13.77 0.03 -0.17
13.79 0.20 0.08
13.81 -0.17 0.07
13.84 0.15 -0.20
13.86 -0.12 0.16
13.89 0.00 -0.22
13.91 0.19 -0.14
13.93 -0.12 0.15
13.96 0.07 -0.31
13.98 0.02 0.07
14.01 0.10 0.10
14.03 0.00 0.24
14.05 0.18 -0.39
14.08 -0.08 0.32
14.10 -0.04 -0.11
14.13 -0.15 -0.17
14.15 -0.18 -0.26
14.17 0.15 -0.11
14.20 0.00 0.23
14.22 -0.19 0.39
14.25 -0.14 0.12
14.27 0.13 0.37
14.29 0.01 0.47
14.32 -0.09 0.85
14.34 0.33 0.97
14.37 0.18 1.73
14.39 0.65 2.07
14.41 0.92 2.81
14.44 1.61 3.61
14.46 2.56 4.83
14.49 3.95 5.86
14.51 5.78 7.80
14.53 8.70 9.62
14.56 12.19 11.65
14.58 17.27 13.71
14.61 23.22 16.31
14.63 30.75 19.22
14.65 39.16 21.21
14.68 48.40 24.35
14.70 58.13 26.80
14.73 67.83 28.88
14.75 76.94 31.51
14.77 84.94 32.86
14.80 90.78 33.98
14.82 94.07 34.83
14.85 95.20 34.81
14.87 93.11 34.84
14.89 88.61 34.25
14.92 81.84 32.51
14.94 73.54 31.18
14.97 64.28 29.91
14.99 54.65 28.31
15.02 45.39 27.15
15.04 36.74 26.37
15.06 29.31 26.30
15.09 23.31 27.59
15.11 18.60 29.29
15.14 14.85 32.40
15.16 12.24 37.32
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15.21 9.92 48.80
15.23 9.85 55.12
15.26 9.50 61.48
15.28 9.94 67.53
15.30 9.92 72.08
15.33 9.95 75.20
15.35 10.25 76.03
15.38 10.08 75.04
15.40 9.57 71.51
15.42 9.36 66.80
15.45 8.75 60.38
15.47 8.00 53.11
15.50 7.17 45.22
15.52 6.24 37.54
15.54 5.53 29.93
15.57 4.41 23.39
15.59 3.64 17.80
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24.00 -0.05 0.02


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README: 50
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Report made with rmarkdown (Allaire, Xie, McPherson, et al., 2021; Xie, Allaire, and Grolemund, 2018; Xie, Dervieux, and Riederer, 2020), RefManageR(McLean, 2020).

Theme is Minty by Bootswatch.


5 Bibliography

Packages

Allaire, J., Y. Xie, J. McPherson, et al. (2021). rmarkdown: Dynamic Documents for R. R package version 2.11. URL: https://CRAN.R-project.org/package=rmarkdown.

McLean, M. W. (2020). RefManageR: Straightforward BibTeX and BibLaTeX Bibliography Management. R package version 1.3.0. URL: https://github.com/ropensci/RefManageR/.

Xie, Y., J. Allaire, and G. Grolemund (2018). R Markdown: The Definitive Guide. ISBN 9781138359338. Boca Raton, Florida: Chapman and Hall/CRC. URL: https://bookdown.org/yihui/rmarkdown.

Xie, Y., C. Dervieux, and E. Riederer (2020). R Markdown Cookbook. ISBN 9780367563837. Boca Raton, Florida: Chapman and Hall/CRC. URL: https://bookdown.org/yihui/rmarkdown-cookbook.

References

d’Adda di Fagagna, F., G. R. Weller, A. J. Doherty, et al. (2003). “The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku”. En. In: EMBO reports 4.1, pp. 47-52.